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Description of key information

In a Buehler test and in a Guinea Pig Maximisation test performed according to OECD Guideline 406, the test substance was observed not to be sensitising to the skin (Armondi, 1990 and BASF 1998).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Murine Local Lymph Node Assay (LLNA) is the first-choice method for in vivo testing according to the REACH Regulation. However, this test was performed before entry into force of the REACH Regulation.
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Dimethylpiperazin
- Physical state: liquid, slight yellowish
- Analytical purity: 99.7 %
- Lot/batch No.: 32-1659
- Storage condition of test material: room temperature, exclusion of oxygen (stored under nitrogen)
Species:
guinea pig
Strain:
other: Pirbright White, Dunkin Hartley Cr1: (HA)BR [SPF]
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH - Wiga, Kisslegg, FRG
- Age at study initiation: Young adult animals
- Weight at study initiation: 348 - 396 g
- Housing: 5 per cage
- Diet (ad libitumn): Kliba Labordiaet 341 (Kaninchen-Meerschweinchen-Haltungsdiaet)
- Water (ad libitum): tap water; about 2 g of ascorbic acid per 10 L water was added to the drinking water twice a week
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Intradermal induction: test substance 0.5% in 0.9% aqueous NaCl-solution
Percutaneous induction: test substance 10% in aqua bidest.
Challenge: test substance 2% in aqua bidest.
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Intradermal induction: test substance 0.5% in 0.9% aqueous NaCl-solution
Percutaneous induction: test substance 10% in aqua bidest.
Challenge: test substance 2% in aqua bidest.
No. of animals per dose:
Test groups: 10
Control groups: 5
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 6 intradermal injections in groups of two per animal ((A) front row: 2 injections each of 0.1 ml Freund's adjuvant* without test substance emulsified with 0.9% aqueous NaCl-solution in a ratio of 1 : 1 (B) middle row: 2 injections each of 0.1 ml of the test substance formulation
(C) back row: 2 injections each of 0.1 ml Freund's adjuvant / 0.9% aqueous NaC1-solution (1 : 1) with test substance) and one percutaneous exposure 1 week after intradermal induction.
- Exposure period: percutaneous exposure: 48 h
- Test groups: 10 animals
- Control group: 5 animals
- Site: shoulder, same area as in the case of the previous intradermal application

B. CHALLENGE EXPOSURE
- No. of exposures: single percutaneous exposure
- Day(s) of challenge: 24 h
- Site: intact flank
- Concentrations: 2 x 2 cm filter paper strips containing the test substance.
- Evaluation (hr after challenge): 24 and 48 h after removal of the patch.

Positive control substance(s):
yes
Remarks:
Performed in a separate study twice a year in the laboratory with alpha-hexylcinnamaldehyde.
Positive control results:
A positive control (reliability check) with a known sensitizer is not included in this study. However, a seperate study is performed twice a year in the laboratory. The positive control with Alpha-Hexylcinnamaldehyde techn. 85% showed that the test system was able to detect sensitizing compounds under the laboratory conditions shown.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
5%
No. with + reactions:
11
Total no. in group:
18
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
5%
No. with + reactions:
5
Total no. in group:
18
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
positive control
Dose level:
5%
No. with + reactions:
8
Total no. in group:
18
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
5%
No. with + reactions:
5
Total no. in group:
118

Maximization Test: Test group results after challenge:

 Form of application: right flank: 2% in aqua bidest.                               
Animal No.  1  2  3  4  5  6  7  8  10
Findings 24 h after removal of the patch  0/0  0/0  0/0  0/0  0/0  0/0  0/0  0/0  0/0  0/0
Findings 48 h after removal of the patch  0/0  0/0  0/0  0/0  0/0  0/0  0/0 0/0   0/0 0/0

x/y: Erythema/Edema

The intradermal induction with 0.5% test substance preparations caused slight to well-defined signs of skin irritation in all test group animals. After the percutaneous induction with a 10% test substance preperation incrustation, partially open (caused by the intradermal induction) could be observed in addition to well-defined erythema and slight edema in all test group animals.

After the challenge with a 2% test substance preparation the animals of control group1 (no application on animals of control group 2) and the test group animals did not show any skin reactions 24 and 48 hours after removal of the patches.

Interpretation of results:
GHS criteria not met
Conclusions:
The intradermal induction with dimethyl piperazine with 0.5% test substance preparations caused slight to well-defined signs of skin irritation in all test group animals. After the percutaneous induction with a 10% test substance preperation incrustation, partially open (caused by the intradermal induction) could be observed in addition to well-defined erythema and slight edema in all test group animals. After the challenge with a 2% test substance preparation the animals of control group 1 (no application on animals of control group 2) and the test group animals did not show any skin reactions 24 and 48 hours after removal of the patches. Based on these results and according to the criteria laid down in the CLP Regulation, the substance is considered not classified as skin sensitizer.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990-09-25 - 1990-11-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
The Murine Local Lymph Node Assay (LLNA) is the first-choice method for in vivo testing according to the REACH Regulation. However, this test was performed before entry into force of the REACH Regulation.
Specific details on test material used for the study:
- Name of test material (as cited in study report): 6398-17-20
- Substance type: Clear, pale yellow viscous liquid
- Physical state: liquid
- Analytical purity: responsibility of the sponsor
- Stability under test conditions: no apparent change in the physical appearance of the test article during administration
- Storage condition of test material: responsibility of the sponsor
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Buckberg Lab Animals, Tomkins Cove, New York
- Weight at study initiation: 300-500g
- Housing: individually in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Purina guinea pig diet, ad libitum
- Water (e.g. ad libitum): fresh tap water, ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


Route:
epicutaneous, occlusive
Vehicle:
other: 80% ethanol (induction) and acetone (challenge)
Concentration / amount:
induction and challenge 0.3 ml/site (25%)
rechallenge 0.3 ml/site (5%)
Route:
epicutaneous, occlusive
Vehicle:
other: 80% ethanol (induction) and acetone (challenge)
Concentration / amount:
induction and challenge 0.3 ml/site (25%)
rechallenge 0.3 ml/site (5%)
No. of animals per dose:
dose range: 8
test article: 20 (10 male, 10 female)
positive control: 5
negative control: 10
naive: 10
Details on study design:
RANGE FINDING TESTS:
2 males, 2 females: each animal is exposed to 4 different concentrations of the test material (1%, 10% 25%, 50%): 80% ethanol as the vehicle.
2 males, 2 females: each animal is exposed to 4 different concentrations of the test material (1%, 10% 25%, 50%): acetone as the vehicle
Primary challenge responses were graded
Highest non-irritatting concentration = concentration that induced responses not exceeding 2 '+' and 2 '0' grades in the group of 4 animals.
Dose chosen for induction and challenge: 25%
Dose chosen for rechallenge: 5%


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 5 (3 inductions, 1 challenge, 1 rechallenge)
- Exposure period: three weeks, a total of three six-hour inductions
- Test groups: test substance in vehicle (80% ethanol)
- Control group: vehicle only
- Site: Left shoulder
- Frequency of applications: once a week
- Duration: 6 h
- Concentrations: 25%


B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: day 29 and 36
- Exposure period: -
- Test groups: test substance in vehicle (acetone)
- Control group: vehicle only (left flank), test article (right flank)
- Site: naive site on left side
- Concentrations: 25% (1st), 5% (2nd)
- Evaluation (hr after challenge): 24 and 48h

OTHER:
24h after challenge and rechallenge, all animals were depilated with Neet Cream Hair Remover (Whitehall Laboratories, Inc., New York).The depilatory was placed on the test sites and surrounding areas and left on for no more than 30 minutes. A minimum of 2h after depilation test sites were graded. The grading was repeated 24h later (48h grade).
Challenge controls:
The negative control group was challenged with vehicle on the left flank and test article on the right flank.
7 days after the primary challenge, all test article treated animals, along with an additional group of naive guina pigs were rechallenged.
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene
Positive control results:
Sensitising effects are observed in all 5 animals of the positive control group.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
severity= 1.9
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
severity= 1.9
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
5%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
severity= 0.0
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
5%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
severity= 0.2
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
severity= 0.0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
severity= 0.0
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
other: naive control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
severity= 0.1
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
other: naive control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
severity= 0.1
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.3%
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
severity= 3.0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.3%
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
severity= 2.8
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% of test substance
No. with + reactions:
3
Total no. in group:
9
Clinical observations:
severity = 1.2
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% of test substance
No. with + reactions:
3
Total no. in group:
9
Clinical observations:
severity = 1.2

One animal died during the course of the study. Necropsy of this animal revealed a slightly enlarged left kidney. The death of this animal was considered not to be related to treatment.

Slight patchy erythema was observed in the test article-treated animals and also in the naive animals treated with the test article at a 5.0% concentration.

Interpretation of results:
GHS criteria not met
Conclusions:
Based upon the observations made in the assay, the test article induced and challenged at a 25% concentration, produced irritation. When rechallenged at 5.0% concentration, the test article did not cause delayed contact hypersensitivity in guinea pigs. Based on these results and according to the criteria laid down in the CLP Regulation, the test substance is considered not classified as non-skin sensitizing.
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Two reliable studies (one buehler test and one guinea pig maximisation test) have been performed with the test substance. Armondi (1990) investigated the sensitising properties of the test substance in a Buehler test with male and female Hartley guinea pigs. In order to determine if the test substance is capable of causing delayed contact hypersensitivity, the substance was dermally applied to twenty guinea pigs (ten males and ten females) for a total of three six-hour insult periods. Another group of five guinea pigs (three males and two females) was treated with 1 -chloro-2,4 -dinitrobenzene (DNCB) at a 0.3% concentration for a total of three six-hour insult periods, as positive control. An additional group of ten guinea pigs (five males and five females) was treated with vehicle for a total of three six-hour insult periods (negative control).

Fourteen days after the last induction period, all animals were challenged at a naive site. A positive response was elicited in the animals receiving the positive control article, 1 -chloro-2,4 -dinitrobenzene (DNCB). No response was observed in the negative control animals treated with the vehicle. Positive scores were observed in the negative control animals treated with test article at a 25% concentration. Positive responses, equal to that of the negative control animals, were observed in the test article-treated animals. Based upon these results, a rechallenge was performed at a 5.0% concentration. Seven days following the primary challenge, the test article-treated animals, along with an additional group of ten naive animals, were rechallenged at a naive site. Slight patchy erythema was observed in the test article-treated animals and also in the naive animals treated with the test article at a 5.0% concentration.

Based upon the observations made in this delayed contact hypersensitivity study, the substance induced and challenged at a 25% concentration, produced irritation. When rechallenged at a 5.0% concentration, the test article did not cause delayed contact hypersensitivity in guinea pigs.

BASF (1998) investigated the sensitising properties of the test substance in a guinea pig maximisation test with male and female Dunkin-Hartley guinea pigs. The study examined the skin sensitising effect on guinea pigs (10 animals in the test group and 5 animals in the control group). Positive and negative controls were included in the study. Animals were induced with 0.5% test substance in 0.9% aqueous NaCl-solution (intradermal induction) and 10% test substance in aqua bidest. (percutaneous induction) and challenged with 2% in aqua bidest. Based upon the observations made in the assay, the substance did not cause skin sensitisation in guinea pigs.

No in vitro skin sensitisation data is available.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the study results and the criteria of the CLP Regulation, the test substance is considered not to be classified as skin sensitising substance.